Characterization of Mycobacterium tuberculosis Isolates from Patients in Houston, Texas, by Spoligotyping

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2000, p Vol. 38, No /00/$ Copyright 2000, American Society for Microbiology. All Rights Reserved. Characterization of Mycobacterium tuberculosis Isolates from Patients in Houston, Texas, by Spoligotyping HANNA SOINI, XI PAN, AMOL AMIN, EDWARD A. GRAVISS, ANEES SIDDIQUI, AND JAMES M. MUSSER* Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, Texas Received 25 August 1999/Returned for modification 27 October 1999/Accepted 9 November 1999 Mycobacterium tuberculosis isolates (n 1,429) from 1,283 patients collected as part of an ongoing population-based tuberculosis epidemiology study in Houston, Texas, were analyzed by spoligotyping and profiling. The isolates were also assigned to one of three major genetic groups on the basis of nucleotide polymorphisms located at codons 463 and 95 in the genes (katg and gyra) encoding catalase-peroxidase and the A subunit of DNA gyrase, respectively. A total of 225 spoligotypes were identified in the 1,429 isolates. There were 54 spoligotypes identified among 713 isolates (n 623 patients) assigned to 73 clusters. In addition, among 716 isolates (n 660 patients) with unique profiles, 200 spoligotypes were identified. No changes were observed either in the profile or in the spoligotype for the 281 isolates collected sequentially from 133 patients. Five instances in which isolates with slightly different spoligotypes had the same profile were identified, suggesting that in rare cases isolates with different spoligotypes can be clonally related. Spoligotypes correlated extremely well with major genetic group designations. Only three very similar spoligotypes were shared by isolates from genetic groups 2 and 3, and none was shared by group 1 and group 2 organisms or by group 1 and group 3 organisms. All organisms belonging to genetic groups 2 and 3 failed to hybridize with spacer probes 33 to 36. Taken together, the results support the existence of three distinct genetic groups of M. tuberculosis organisms and provide new information about the relationship between profiles, spoligotypes, and major genetic groups of M. tuberculosis. * Corresponding author. Present address: Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th St., Hamilton, MT Phone: (406) Fax: (406) jmusser@niaid.nih.gov. Spacer oligonucleotide typing (spoligotyping) is a molecular method used to differentiate Mycobacterium tuberculosis complex isolates. This method is based on the analysis of polymorphisms in the M. tuberculosis complex direct repeat (DR) chromosomal region consisting of identical 36-bp DRs alternating with 35- to 41-bp unique spacer sequences. The method is PCR based and hence is more rapid and easier to perform than the standard typing technique based on profiling (10, 14). Spoligotyping can also be performed directly from M. tuberculosis organisms, even those that are nonviable or that are found in tissues in paraffin-embedded blocks, or in archeological samples (7, 19, 23). Several studies have provided evidence that spoligotyping is less able to discriminate among isolates with high copy numbers, whereas spoligotyping is superior to profiling for isolates with fewer than five copies (3, 9, 14, 16, 27). Thus, a two-step protocol consisting of initial screening of isolates by spoligotyping, followed by profiling of isolates with the same spoligotype, has been suggested (8). Spoligotyping also has been reported to be a useful method for the differentiation of Mycobacterium bovis isolates, because the majority of isolates of this species have only one element. In addition, the absence of spacers 3, 9, 16, and 39 to 43 is characteristic of M. bovis isolates (2, 5, 6, 11, 29). Similarly, Mycobacterium microti and Mycobacterium canettii have characteristic spoligotypes (15, 18, 26). For accurate interpretation of spoligotype data, it is necessary to obtain information about the evolution and relative stability of the DR region in large samples of isolates from diverse geographic sources. It is also necessary to gain insight into such issues as the relationship of spoligotypes to the three principal genetic groups of M. tuberculosis (22) and the likelihood of evolutionary convergence to the same spoligotype. To address these and other issues, we studied the relationship between spoligotype, profile, and principal genetic group in 1,429 M. tuberculosis isolates causing disease in Houston, Texas. MATERIALS AND METHODS Bacterial isolates. The analysis is based on 1,429 M. tuberculosis isolates collected from 1,283 patients as part of an ongoing, population-based tuberculosis epidemiology study in Houston, Texas. The strains were cultured from patients between September 1994 and February These organisms included 281 isolates recovered sequentially from 133 patients. DNA methods. Chromosomal DNA extraction and profiling were performed by an internationally standardized protocol (24). The profiles were analyzed with the BioImage (Ann Arbor, Mich.) Whole Band Analysis program, version 3.2. Spoligotyping was performed with a commercially available kit (Isogen Bioscience BV, Maarssen, The Netherlands) according to the instructions supplied by the manufacturer. The isolates were assigned to one of three principal genetic groups on the basis of nucleotide polymorphism at codons 463 and 95 of the genes encoding catalase-peroxidase and the A subunit of DNA gyrase, respectively (22). Statistical analysis. Statistical analysis was performed with Epi Info, version 6.04 (Centers for Disease Control and Prevention, Atlanta, Ga.). Chi-square analysis was used to test the association of clustered spoligotypes with clustering and with the three major genetic groups. The sensitivity and specificity of spoligotyping were calculated by a method described by Hennekens and Buring (12), by using clustering by as the gold standard. The sensitivity was calculated as the number of patients clustered by both spoligotyping and profiling, divided by the total number of patients clustered by profiling. The specificity was calculated as the number of patients clustered neither by spoligotyping nor by profiling, divided by the total number of patients not clustered by profiling. RESULTS Spoligotype and type. A sample of 1,429 M. tuberculosis isolates from 1,283 patients was analyzed by pro- 669

2 670 SOINI ET AL. J. CLIN. MICROBIOL. FIG. 1. Spoligotypes identified in Houston M. tuberculosis isolates. Column heads: Spoligotype, arbitrary spoligotype designation; numbers 1 to 43, spoligotype probes (an x in the field below denotes hybridization, and an empty square indicates lack of hybridization); Group, major genetic group designation; Patients, number of patients infected by an isolate with this spoligotype. Boldfaced numbers in parentheses following arbitrary spoligotype designations correspond to the spoligotype designations provided by Sola et al. (21). filing and spoligotyping. An isolate was assigned a print designation if the same pattern was found for isolates obtained from two or more patients. If no matching profiles were identified in the database, or if the pattern contained fewer than 5 copies, the isolate was defined as unique (print 999) (exception: print 006 has a copy number of 4). A total of 225 spoligotypes were identified in the 1,429 isolates (Fig. 1). There were 54 spoligotypes identified among 713 isolates (n 623 patients) assigned to 73 clusters. In addition, among 716 isolates (n 660 patients) with unique profiles, 200 spoligotypes were identified. Twenty-nine spoligotypes were shared by clustered and unique isolates. By spoligotype alone, isolates from 1,146 patients were divided into 89 spoligotypes, whereas 137 patients were infected

3 VOL. 38, 2000 SPOLIGOTYPING OF M. TUBERCULOSIS ISOLATES 671 FIG. 1 Continued. by organisms with unique spoligotypes. Patients with clustered spoligotypes (the same spoligotype identified in multiple patients) were significantly associated with clustering ( ; P 0.001). Although the sensitivity of the spoligotyping technique was fairly high (599 of 623; 96%), the specificity of spoligotyping in differentiating -clustered clones from nonclustered clones was relatively low (113 of 660; 17%). Spoligotype and major genetic group. All isolates were also assigned to one of the three major genetic groups. In general, there was little sharing of major genetic groups and spoligotypes. Moreover, there was no association between the major genetic groups and spoligotype clustering (P 0.19). We identified three very similar spoligotypes that were shared by group 2 and group 3 organisms (Table 1), but no sharing of spoligotypes among organisms belonging to major genetic groups 1 and 2, or among group 1 and group 3 organisms, was found. In addition, all isolates of major genetic groups 2 and 3 failed to hybridize with spoligotype probes 33 to 36. These results are consistent with the genetic affiliation of group 2 and group 3

4 672 SOINI ET AL. J. CLIN. MICROBIOL. FIG. 1 Continued. organisms and the differentiation of group 1 and group 3 organisms (22). Analysis of large clusters. We next examined spoligotype variation among isolates classified on the basis of profile. To maximize the opportunity to identify spoligotype variation among isolates assigned to an profile, we studied seven large clusters with 40 to 123 isolates each. In general, isolates in each cluster had the same spoligotype. However, we identified two instances in which an isolate with a slightly different spoligotype had the same profile. In the case of print 006, which has 4 copies, 10 different spoligotypes were obtained. In addition, an isolate with a different spoligotype was observed in three print groups with few isolates (005, 085, and 146) (Table 2). The more-abundant spoligotypes. Seventeen of the 23 types in genetic group 1 had the same spoligotype, arbitrarily designated S1. This spoligotype was characterized by hybridization with probes 35 to 43 and has been identified previously

5 VOL. 38, 2000 SPOLIGOTYPING OF M. TUBERCULOSIS ISOLATES 673 FIG. 1 Continued. (25). Spoligotype S1 was identified in all 309 isolates from 264 patients infected with these 17 types and was also identified in 67 isolates (62 patients) with unique profiles. The most common spoligotype found among isolates with low copy numbers was S12. This pattern was obtained from 147 isolates (from 134 patients) with copy numbers ranging from 2 to 5 and has been identified previously (3) (Table 3). Serial isolates. Our study included 281 isolates collected sequentially from 133 patients. The interval between isolate collections varied from 1 to 1,043 days (average, 87 days). Although 76% of the organisms were obtained within 60 days of one another, no changes were observed either in the profile or in the spoligotype. DISCUSSION Correlation of spoligotypes with major genetic groups. Recently three genetic groups of M. tuberculosis isolates were identified on the basis of polymorphic nucleotides in katg codon 463 and gyra codon 95 (22). According to the evolu-

6 674 SOINI ET AL. J. CLIN. MICROBIOL. TABLE 1. Spoligotypes shared by major genetic groups Spoligotype designation a Spoligotype pattern profile a copy no. isolates patients Genetic group b S29 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx...xxxxxxx c S91 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx...xxx.xxx d S77 xxxxxxxxxxxx.xxxxxxxxxxxxxxxxxxx...xxxxxxx e a Arbitrary designation. b Defined by Sreevatsan et al. (22). c Spoligotype 53 defined by Sola et al. (21). d Spoligotype 52 defined by Sola et al. (21). e Spoligotype 37 defined by Sola et al. (21). tionary scenario proposed in that study, group 1 isolates are evolutionarily old and have further evolved into group 2 and group 3 organisms (22). Our results show that spoligotypes correlate extremely well with major genetic group designations. Only three very similar spoligotypes were shared by group 2 and group 3 organisms, and, as anticipated, none was shared between group 1 and group 3 organisms. The three shared spoligotypes have been reported to be globally distributed (21). It is probable that the group 3 isolates evolved from a group 2 precursor isolate. Consistent with this idea, we observed that all isolates belonging to major genetic groups 2 and 3 failed to hybridize with spacer probes 33 to 36, suggesting that these spacers and DRs have been deleted from the genomes of all group 2 and group 3 organisms. Taken together, these results further support the division of M. tuberculosis isolates into three distinct genetic groups. Stability of spoligotypes. Relatively little is known about the molecular evolution and stability of the DR region in M. tuberculosis complex isolates. In a recent study, Niemann et al. profile a copy no. Genetic group b TABLE 2. profiles characterized by different spoligotypes isolates patients Spoligotype designation a analyzed drug-resistant M. tuberculosis isolates recovered sequentially from 56 patients (17). They found no change in the spoligotypes obtained from these isolates, whereas in five cases a change in the profile was observed (addition or deletion of one band) (17). Similarly, in other studies that included duplicate or serial isolates from the same patient, the spoligotypes were identical (7 9, 13, 20). No change in spoligotypes or profiles was observed in the serial isolates included in our study. Although the interval between collections of sequential isolates varied from 1 to 1,043 days, 76% of the organisms were obtained within 60 days of one another, which may be too short a period for variation to arise in this region. Our study also included clusters consisting of a large number of M. tuberculosis isolates with identical or very similar profiles. We identified only five cases in which an apparent change in DR pattern occurred. These results suggest that in rare cases, isolates with different spoligotypes can be clonally related. Spoligotype pattern S24 xxxxxxxx...xxxxxxxxxxxxxxxxxxx...xxxxxxx S245 xxxxxxxx...x...xxxxxxxxxxxxxx...xxxxxxx S3 xxxxxxxxxxxxxxxxx.xxxxxxxxxxxxxx...xxxxxxx S5 xxxxxxxxxxxx.xxx...xxxxxxxxxxxxx...xxxxxxx H37Rv xxxxxxxxxxxxxxxxxxx..xxxxxxxxxxx...xxxxxxx S83 xxxxxxxxxxxxxxxxxxx..xxxxxxxxxxx...xx..xxx S25 xxx...x...xxxxxxxxxxxxxx...xxxxxxx S33 xxx...xxxxx.xxxxxxxxxxxxxx...xxx S256 xxx...xxxxx.xxxxxxxxxxxxxx...xxxxxx S27 xxx...xxxxx.xxxxxxxxxxxxxx...xxxxxxx S26 xxxxxxxxxxxxx...xxxxxxxxxxxxxx...xxxxxxx S30 xxxxxxxxxxxxx...xxxxxxxxxxxxxxx...xxxxxxx S31 xxxxxxxxxxxxxx.xx.xxxxxxxxxxxxxx...xxxxxxx S3 xxxxxxxxxxxxxxxxx.xxxxxxxxxxxxxx...xxxxxxx S32 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx...xxxxxxx S29 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx...xxxxxxx S78 xxxxxxxxxxxxxxxxxxx.xx.xxxxxxxxx...xxxxxxx S100 xxxxxxxxxxxxxxxx...x.xxxxxxxxx...xxxxxxx S28 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxx.x...xxxxxxx S206 xxxx.xxxxxxxxxxxxxxxxxxxxxxxxx.x...xxxxxxx a Arbitrary designation. b Defined by Sreevatsan et al. (22).

7 VOL. 38, 2000 SPOLIGOTYPING OF M. TUBERCULOSIS ISOLATES 675 TABLE 3. The most common spoligotypes observed in the Houston area Spoligotype designation a Spoligotype pattern profile a copy no. isolates patients Genetic group b S1...xxxxxxxxx c S12 xxxxxxxxxxxxxxxxx.xxxxxxxxxxxxxx...xx...x a Arbitrary designation. b Defined by Sreevatsan et al. (22). c Spoligotype 1 defined by Sola et al. (21). Correlation with published spoligotypes. On the basis of published spoligotypes, Sola et al. (21) compiled a table of 69 spoligotypes shared by more than two patients in any region of the world. Twenty-six of the 69 spoligotypes were also identified in Houston (Fig. 1). Four of these (spoligotypes 17, 29, 44, and 67) were spoligotypes previously identified in the Caribbean and neighboring Central American regions only. One of the Houston patients infected by an organism with a Caribbean-specific spoligotype was born in the Caribbean (St. Lucia), but no direct epidemiologic links could be found for the other patients. A large number of the Houston patients (326 of 1283; 25%) were infected by an M. tuberculosis isolate of spoligotype S1. This is the characteristic pattern of the Beijing family of M. tuberculosis isolates, which is prevalent in China and neighboring countries but uncommon in Europe and the Caribbean (21, 25). This is also the spoligotype of the W family of M. tuberculosis isolates, which is a group of closely related multidrugresistant organisms that have recently spread from New York City to other U.S. communities and to Paris (1, 4). Our results indicate that M. tuberculosis isolates with this spoligotype are also common in Houston. In contrast to spoligotype S1, the majority of the spoligotypes identified in Houston have not been described previously. Our spoligotype data thus support the results obtained by profiling showing that most M. tuberculosis isolates are confined to a specific geographic location (28). ACKNOWLEDGMENT This research was supported by Public Health Service grant DA to J.M.M. REFERENCES 1. Agerton, T. B., S. E. Valway, R. J. Blinkhorn, K. L. Shilkret, R. Reves, W. W. Schluter, B. Gore, C. J. Pozsik, B. B. Plikaytis, C. Woodley, and I. M. Onorato Spread of strain W, a highly drug-resistant strain of Mycobacterium tuberculosis, across the United States. Clin. Infect. Dis. 29: Aranaz, A., E. Liebana, A. Mateos, L. Dominguez, D. Vidal, M. Domingo, O. Gonzolez, E. F. Rodriguez-Ferri, A. E. Bunschoten, J. D. A. van Embden, and D. Cousins Spacer oligonucleotide typing of Mycobacterium bovis strains from cattle and other animals: a tool for studying epidemiology of tuberculosis. J. Clin. Microbiol. 34: Bauer, J., A. B. Andersen, K. Kremer, and H. Miorner Usefulness of spoligotyping to discriminate low-copy-number Mycobacterium tuberculosis complex strains cultured in Denmark. J. Clin. Microbiol. 37: Bifani, P. J., B. B. Plikaytis, V. Kapur, K. Stockbauer, X. Pan, M. L. Lutfey, S. L. Moghazeh, W. Eisner, T. M. Daniel, M. H. Kaplan, J. T. Crawford, J. M. Musser, and B. N. Kreiswirth Origin and interstate spread of a New York City multidrug-resistant Mycobacterium tuberculosis clone family. JAMA 275: Blazquez, J., L. E. Espinosa de los Monteros, S. Samper, C. Martin, A. Guerrero, J. Cobo, J. van Embden, F. Baquero, and E. Gomez-Mampaso Genetic characterization of multidrug-resistant Mycobacterium bovis strains from a hospital outbreak involving human immunodeficiency viruspositive patients. J. Clin. Microbiol. 35: Cousins, D., S. Williams, E. Liebana, A. Aranaz, A. Bunschoten, J. van Embden, and T. Ellis Evaluation of four DNA typing techniques in epidemiological investigations of bovine tuberculosis. J. Clin. Microbiol. 36: Driscoll, J. R., M. A. McGarry, and H. W. Taber DNA typing of a nonviable culture of Mycobacterium tuberculosis in a homeless shelter outbreak. J. Clin. Microbiol. 37: Goguet de la Salmonière, Y.-O., H. M. Li, G. Torrea, A. Bunschoten, J. van Embden, and B. Gicquel Evaluation of spoligotyping in a study of the transmission of Mycobacterium tuberculosis. J. Clin. Microbiol. 35: Goyal, M., N. A. Saunders, J. D. A. van Embden, D. B. Young, and R. J. Shaw Differentiation of Mycobacterium tuberculosis isolates by spoligotyping and restriction fragment length polymorphism. J. Clin. Microbiol. 35: Groenen, P. M. A., A. E. Bunschoten, D. van Soolingen, and J. D. A. van Embden Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method. Mol. Microbiol. 10: Gutierrez, M., S. Samper, M. S. Jimenez, J. D. A. van Embden, J. F. G. Marin, and C. Martin Identification by spoligotyping of a caprine genotype in Mycobacterium bovis strains causing human tuberculosis. J. Clin. Microbiol. 35: Hennekens, C. H., and J. E. Buring Epidemiology in disease control, p In S. L. Mayrent (ed.), Epidemiology in medicine. Little, Brown & Co., Boston, Mass. 13. Horgen, L., C. Sola, A. Devallois, K. S. Goh, and N. Rastogi Follow up of Mycobacterium tuberculosis transmission in the French West Indies by -DNA fingerprinting and DR-based spoligotyping. FEMS Immunol. Med. Microbiol. 21: Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, S.

8 676 SOINI ET AL. J. CLIN. MICROBIOL. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. van Embden Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35: Kremer, K., D. van Soolingen, J. van Embden, S. Hughes, J. Inwald, and G. Hewinson Mycobacterium microti: more widespread than previously thought. J. Clin. Microbiol. 36: Kremer, K., D. van Soolingen, R. Frothingham, W. H. Haas, P. W. M. Hermans, C. Martin, P. Palittapongarnpim, B. B. Plikaytis, L. W. Riley, M. A. Yakrus, J. M. Musser, and J. D. A. van Embden Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. J. Clin. Microbiol. 37: Niemann, S., E. Richter, and S. Rusch-Gerdes Stability of Mycobacterium tuberculosis restriction fragment length polymorphism patterns and spoligotypes determined by analyzing serial isolates from patients with drug-resistant tuberculosis. J. Clin. Microbiol. 37: Pfyffer, G. E., R. Auckenthaler, J. D. A. van Embden, and D. van Soolingen Mycobacterium canettii, the smooth variant of M. tuberculosis, isolated from a Swiss patient exposed in Africa. Emerg. Infect. Dis. 4: Qian, L., J. D. A. van Embden, A. G. M. van der Zanden, E. F. Weltevreden, H. Duanmu, and J. T. Douglas Retrospective analysis of the Beijing family of Mycobacterium tuberculosis in preserved lung tissues. J. Clin. Microbiol. 37: Sola, C., L. Horgen, J. Maisetti, A. Devallois, K. S. Goh, and N. Rastogi Spoligotyping followed by double-repetitive-element PCR as rapid alternative to fingerprinting for epidemiological studies of tuberculosis. J. Clin. Microbiol. 36: Sola, C., A. Devallois, L. Horgen, J. Maisetti, I. Filliol, E. Legrand, and N. Rastogi Tuberculosis in the Caribbean: using spacer oligonucleotide typing to understand strain origin and transmission. Emerg. Infect. Dis. 5: Sreevatsan, S., X. Pan, K. E. Stockbauer, N. D. Connell, B. N. Kreiswirth, T. S. Whittam, and J. M. Musser Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc. Natl. Acad. Sci. USA 94: Taylor, G. M., M. Goyal, A. J. Legge, R. J. Shaw, and D. Young Genotypic analysis of Mycobacterium tuberculosis from medieval human remains. Microbiology 145: van Embden, J. D. A., M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J. Clin. Microbiol. 31: van Soolingen, D., L. Qian, P. E. W. de Haas, J. T. Douglas, H. Traore, F. Portaels, H. Z. Qing, D. Enkhsaikan, P. Nymadwa, and J. D. A. van Embden Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia. J. Clin. Microbiol. 33: van Soolingen, D., A. G. M. van der Zanden, P. E. W. de Haas, G. T. Noordhoek, A. Kiers, N. A. Foudraine, F. Portaels, A. H. J. Kolk, K. Kremer, and J. D. A. van Embden Diagnosis of Mycobacterium microti infections among humans by using novel genetic markers. J. Clin. Microbiol. 36: Wilson, S. M., S. Goss, and F. Drobnievski Evaluation of strategies for molecular fingerprinting for use in the routine work of a mycobacterium reference unit. J. Clin. Microbiol. 36: Yang, Z., P. F. Barnes, F. Chaves, K. D. Eisenach, S. E. Weis, J. H. Bates, and M. D. Cave Diversity of DNA fingerprints of Mycobacterium tuberculosis isolates in the United States. J. Clin. Microbiol. 36: Zumarraga, M. J., C. Martin, S. Samper, A. Alito, O. Latini, F. Bigi, E. Roxo, M. E. Cicuta, F. Errico, M. C. Ramos, A. Cataldi, D. van Soolingen, and M. I. Romano Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis-related infections in South America. J. Clin. Microbiol. 37: Downloaded from on September 1, 2018 by guest